Nina Baluja, M.D.

	On the heels of the 2017 FDA approvals of Kymriah® (tisagenlecleucel) to treat leukemia and Yescarta® (axicabtagene ciloleucel) to combat large-B-cell lymphomas, researchers are continuing to make bold strides in revolutionizing cancer therapy. While some patients experience potent and durable clinical responses to these breakthrough immunotherapies, response rates are highly variable, and these treatments are often associated with side effects that differ from those of cytotoxic chemotherapy. As a result, researchers and clinicians are in search of tools that help them identify which patients are most likely to benefit from specific immunotherapies.

	There is a critical need for standardized and validated biomarkers that yield actionable insights into immunotherapy efficacy at every stage of development. In addition to helping identify patients who could benefit from available therapies, biomarkers may be useful for monitoring treatment response. These indicators also have the potential to shed light on a treatment’s mechanism of action, which would provide important insight for optimizing treatment approaches and defining rational combination therapies. However, the intrinsic characteristics of malignant tumors-such as their heterogeneity, plasticity, and diversity-pose challenges to biomarker development.

	Here are four trends to watch as researchers continue to make headway in the development of biomarkers for cancer immunotherapy.

Using state of the art technologies for biomarker development

	Only a tiny fraction of the tens of thousands of identified biomarkers have been developed into validated genomic biomarkers for FDA-approved drugs, and none have become in vitro companion diagnostics.

 To be used effectively in a clinical setting, predictive biomarkers must have both clinical utility and analytic and clinical validity. Many organizations have published guidelines on validating diagnostic tests, which provide recommendations on analytic sensitivity, specificity, reproducibility, and assay robustness.2,3

	The translation of biological data into predictive or prognostic biomarkers is complicated by the many host-and cancer-related factors that influence the complex interactions between tumors and the immune system. New genomic and proteomic technologies, combined with advanced bioinformatic tools, enable the simultaneous analysis of thousands of biological molecules. These cutting-edge techniques help enable the discovery of new tumor signatures, which are critical for making the leap to precision medicine and personalized cancer therapy.

	Mass cytometry, whole-exome sequencing, gene expression profiling, and T-cell receptor clonality sequencing technology are just a few of the novel technologies and high-throughput approaches being used for biomarker development. With these techniques, a single sample can be used to address many questions, but the resulting quantity and complexity of data leads to unique analytical challenges and may require multi-disciplinary expertise for interpretation.

Validating PD-L1 as a biomarker of response

	Programmed death ligand-1 (PD-L1) a transmembrane protein expressed on a variety of cell types, including dendritic cells. The main programmed death ligand among the immune checkpoint inhibitory receptors known as PD-1s, PD-L1 plays a critical role in innate and adaptive immunity. PD-L1 binding inhibits the normal function of activated T-cells. By expressing PD-L1, tumor cells can co-opt the PD-1/PD-L1 regulatory mechanism and inhibit T-cell activation, allowing cancer cells to bypass the immune system.

	Therapeutic antibodies can be used to block PD-1 or PD-L1 and restore host anti-tumor immunity by removing the suppressive effects of PD-L1 on cytotoxic T-cells. As such, defining biomarkers that predict therapeutic response to PD-1/PD-L1 blockade is critical for identifying patients who are likely to respond to PD-1 and PD-L1 immune checkpoint inhibitors.  

	In clinical studies, immunohistochemistry has been used to detect PD-L1 protein expression by tumor cells and evaluate it for correlation with response to PD-1 and PD-L1 immune checkpoint inhibitors. Currently, the only FDA-approved companion diagnostic is PD-L1 IHC 22C3 pharmDx, which is used to select patients for treatment with pembrolizumab, a PD-1 inhibitor marketed as Keytruda.

	Unfortunately, PD-L1 negativity is unreliable and results may vary depending on antibody, assay, or tissue sample. Tumor heterogeneity, low expression, and inducible genes can also lead to sampling errors or false negatives. In addition, at this time, it is not known how previous cancer treatments affect the tumor microenvironment.4 According to a recent Blueprint Working Group analysis comparing immunohistochemistry tests and cell scoring methods for PD-L1 expression, more data is needed before an alternative assay can be used to read specific therapy-related PD-L1 cutoffs.5

	For now, PD-L1 IHC positivity remains an imperfect biomarker of response and cannot be used as a definitive biomarker to select patients for PD-1/PD-L1 inhibitor therapy. A more complex, multi-component predictive biomarker system likely will be required to refine patient selection.6

Linking tumor mutation burden to response rate

	Tumor mutation burden (TMB), a measure of the mutations carried by tumor cells, has been studied to evaluate its association with response to immuno-oncology therapy. DNA sequencing is used to determine the number of acquired mutations in a tumor, and TMB is generally reported as the number of mutations in a specific area of genetic material.

	The theory behind TMB as a biomarker is that tumor cells with high TMB may have more neoantigens, cell-surface molecules produced by DNA mutations that are unique to cancer cells. These neoantigens can be recognized by T-cells to stimulate an anti-tumor immune response in the tumor microenvironment. Consequently, a high TMB may correlate positively with the likelihood of responding to immunotherapy.

	At the 2017 World Conference on Lung Cancer, researchers presented data from CheckMate-032, an ongoing Phase I/II open-label trial comparing nivolumab monotherapy with nivolumab plus ipilimumab combination therapy in patients with advanced small-cell lung cancer. The data showed that a high TMB predicted better outcomes, regardless of the treatment arm, compared with medium of low TMB. In addition, patients with a high TMB who received combination therapy had significantly higher response rates and one-year overall survival than those who received monotherapy. Taken together, these findings provide strong support for the clinical utility of TMB as a biomarker for nivolumab therapy, both alone and in combination with ipilimumab.7

Leveraging the tumor microenvironment to direct therapeutic development

	The tumor microenvironment comprises the cellular environment in which a tumor exists, including surrounding vasculature, immune cells, fibroblasts, inflammatory cells, signaling molecules, and the extracellular matrix. Recent investigations into the tumor microenvironment seek to determine whether genetic changes can guide the design of cancer immunotherapeutics. Unlike predictive or prognostic biomarkers, immune targets may not correlate with treatment response, but may help direct the development of new cancer therapies.

	In one study using Ras mutations as immune target biomarkers, 57 patients with advanced solid tumors bearing Ras mutations received a cancer vaccine containing autologous peptides and interleukin-2, granulocyte-macrophage colony-stimulating factor, or both. While most patients developed antigen-specific immune responses, only one generated productive, tumor-eliminating immunity.

 This finding led to the discovery that there is a significant expansion of regulatory T-cells (Tregs) in patients with colon tumors bearing Ras mutations.

	Mutant Ras induces secretion of high levels of IL-10 and transforming growth factor-β1, which generate local induction of Treg in the tumor microenvironment.

Induction of Treg supports tumor immune escape by creating a suppressive tumor microenvironment that inhibits the anti-tumor response, suggesting that the efficacy of cancer vaccines in patients with Ras mutations may improve with addition of an agent that targets Treg.

	Immunotherapies represent a paradigm shift in cancer treatment and are expanding the therapeutic landscape for cancer patients. However, they are not a one-size-fits-all solution and there is growing need to identify predictive and prognostic markers that enhance our understanding of the complex interactions between cancer and the immune system, as well as our ability to identify the right therapy for each patient at every stage of their disease. We are still at the dawn of cancer immunotherapy biomarker development, with exciting opportunities ahead for using biomarkers to aid in treatment selection and rational design of the combination therapies that will be the next revolution in cancer treatment.

	1 Gulley JL, et al. Immunotherapy biomarkers 2016: overcoming the barriers. J Immunother Cancer 2017;5:29.

	2 Chau CH, Rixe O, McLeod H, Figg WD. Validation of analytic methods for biomarkers used in drug development. Clin Cancer Res 2008;14(19);5967-5976.

	3 Lee JW, et al. Method validation and measurement of biomarkers in nonclinical and clinical samples in drug development: a conference report. Pharm Res 2005;22(4):499-511.

	4 Topalian SL, et al. Mechanism-driven biomarkers to guide immune checkpoint blockade in cancer therapy. Nat Rev Cancer 2016;5:275-287.

	5 Hirsch FR, et al. PD-LD Immunohistochemistry assays for lung cancer: Results from Phase 1 of the Blueprint PD-L1 IHC Assay Comparison Project. J Thorac Oncol 2017;12(2):208-222.

	6 Boussiotis VA. Molecular and biochemical aspects of the PD-1 checkpoint pathway. N Engl J Med 2016;375:1767-1778.

	5 Chau CH, Rixe O, McLeod H, Figg WD. Validation of analytic methods for biomarkers used in drug development. Clin Cancer Res 2008;14(19);5967-5976.

	6 Lee JW, et al. Method validation and measurement of biomarkers in nonclinical and clinical samples in drug development: a conference report. Pharm Res 2005;22(4):499-511.

	7 Rizvi N, et al. Impact of tumor mutation burden on the efficacy of nivolumab or nivolumab plus ipilimumab in small cell lung cancer: An exploratory analysis of CheckMate 032. 2017 World Conference on Lung Cancer. Abstract OA 07.03a. Presented October 16, 2017.

	8 Rahma OE, et al. The immunological and clinical effects of mutated ras peptide vaccine in combination with IL-1, GM-CSF, or both in patients with solid tumors. J Trans Med 2014;12:55.

	9 Zdanov S, et al. Mutant KRAS conversion of conventional T cells into regulatory T cells. Cancer Immunol Res 2016;4(4):354-365.

, Senior Medical Director, Premier Research

Articles

Here are four trends to watch as researchers continue to make headway in the development of biomarkers for cancer immunotherapy.

Cancer Immunotherapy Biomarkers: Four Trends to Watch

	Cancer was once considered a disease of genetic origin, with hallmarks including sustained proliferation, resistance to apoptosis, the ability to promote angiogenesis, and the ability to promote invasion and metastasis. However, this view failed to take into consideration the dynamic nature of the interactions between a tumor and its microenvironment-not just the normal cells in the surrounding tissue, but also the immune system.

	Advances in our understanding of the dual role that the immune system plays in cancer have led to the development of immune checkpoint inhibitors, cancer therapies that prevent cancer cells from turning off T cells, enabling those T cells to infiltrate the tumor and stop it from growing. However, many patients with cancer do not respond to treatments with immune checkpoint inhibitors, in part due to the lack of tumor-infiltrating effector T cells.

	Cancer vaccines may help to prime patients for treatments with immune checkpoint inhibitors by inducing both effector T cell infiltration into the tumors and immune checkpoint signals. As such, combination therapy with a cancer vaccine and an immune checkpoint inhibitor may function synergistically to induce more effective anti-tumor immune responses.

	In this article, we will explore the role of the immune system in cancer and discuss the potential of cancer immunotherapies that combine cancer vaccines and immune checkpoint inhibitors.

	To understand how cancer vaccines may be used to potentiate other immunotherapies, it is important to understand the fundamentals of tumor immunology:

	1. To initiate immunity, dendritic cells (DCs) must sample antigens derived from the tumor, which can be ingested in situ or delivered exogenously as part of a therapeutic vaccine, making DCs a potential site for therapeutic intervention. Upon antigen encounter, the DCs must also have to receive a suitable activation (or, maturation) signal, allowing them to differentiate extensively to promote immunity, as opposed to tolerance.

	2. Next, tumor antigen-loaded DCs must generate protective T cell responses. The precise type of T cell response needed is unknown, but must include the production of CD8+ effector T cells with cytotoxic potential. DCs may also trigger antibody and natural killer/natural killer T (NK/NKT) cell responses, which may contribute to tumor immunity.

	3. Finally, cancer-specific T cells must enter the tumor bed to perform their function. Here, there is the challenge of immune suppression. Presumably by skewing DC maturation, tumors may:

		Trigger the “wrong” immune response, or

		Enable the local accumulation or expansion of regulatory T cells (Treg cells) that would oppose the activity of effector T cells.

	Cancer vaccines come in two formats: prophylactic and therapeutic. Prophylactic vaccines have been used with considerable success in preventing cancers of viral origin, such as hepatitis B virus (HBV) and human papillomavirus (HPV), where the etiologic agent is known. In contrast, the development of therapeutic vaccines has been problematic, with many promising phase II studies failing to show survival benefit in phase III trials.

	The hypothesis behind therapeutic cancer vaccines originated with the discovery that patients with cancer can harbor CD8+ and CD4+ T cells specific for cancer/testis antigens or differentiation antigens expressed in their tumors. Vaccination might reasonably be expected to amplify the frequency and strength of these pre-existing responses, or perhaps induce de novo reactions. In addition, clinico-pathologic studies have demonstrated a strong association between prolonged patient survival and the presence of intra-tumoral CD3+ or CD8+ cytotoxic T cells and an interferon-gamma (IFN-γ) gene signature. If vaccination could trigger these types of T cell responses, then clinical benefit might be expected.

	To date, two therapeutic cancer vaccines have been approved by the FDA: sipuleucel-T and talimogene laherparepvec.

	1. Sipuleucel-T (marketed as Provenge®) received FDA approval in April 2010 for the indication of advanced prostate cancer.

 Originally assumed to be an autologous DC-based vaccine, sipuleucel-T is made up of an incompletely characterized, complex mixture of peripheral blood mononuclear cells (PBMCs) supplemented with a cytokine and tumor-derived differentiation antigen.
	
	A phase III trial of sipuleucel-T showed little evidence of tumor shrinkage or delay in disease progression. By standard Response Evaluation Criteria in Solid Tumors (RECIST) criteria, only one of the 341 patients in the active arm exhibited a partial response. However, the study did show a 4.1-month improvement in median overall survival (25.8 months vs 21.7 months). This survival benefit was deemed significant by the FDA in a patient population that has few, if any, other effective therapeutic options.

	2. Talimogene laherparepvec (T-VEC, marketed as Imlygic®) was approved by the FDA in October 2015 for treatment of melanoma lesions in the skin and lymph nodes.

 T-VEC is a genetically modified live oncolytic herpes virus therapy that is injected directly into melanoma lesions, where it replicates inside cancer cells and causes apoptosis. Oncolytic virus therapy mediates tumor regression through two distinct mechanisms. First, many viruses possess an innate tropism for cancer cells where they can preferentially replicate and kill established tumor cells. Secondly, the dying tumor cells can serve as a target for cross priming tumor-specific immune responses to generate systemic anti-tumor immunity.

	The safety and efficacy of T-VEC were evaluated in a multi-center study of 436 patients with metastatic melanoma that could not be surgical removed. The study showed that 16.3 percent of study participants who received T-VEC experienced a decrease in the size of their melanoma lesions, lasting for at least six months, compared to 2.1 percent of study participants who received comparator therapy. However, T-VEC has not been shown to improve overall survival, or to have an effect on melanoma that has metastasized to the brain, bone, liver, lungs, or other internal organs.

	Other oncolytic viruses that may be nearing approval in North American and Europe include pexastimogene devacirepvec (a vaccinia virus) for hepatocellular carcinoma, GM-CSF-expressing adenovirus CG0070 for bladder cancer, and palavered (a wild-type variant of reovirus) for head and neck cancer.

	There are a variety of challenges associated with the development of therapeutic cancer vaccines:

	· The vaccine initially induces an immune reaction against the vaccine itself, not the tumor. As such, the immune system mainly recognizes “neo-antigens” from “passenger” mutations rather than shared antigens.

	· The antigens are different for each tumor. Consequently, a therapeutic cancer vaccine would need to involve autologous tumor cells.

	· Most immune-responsive tumors auto-vaccinate, but immune regulation prevents an effective response. Even if a vaccine enhances anti-tumor immunity, cells are unlikely to be suppressed in the tumor microenvironment.

	Based on the foregoing issues, vaccines are unlikely to have a major anti-tumor effect in the absence of immune checkpoint control.

Immune Checkpoint Inhibitors: A Turning Point in Cancer Immunotherapy

	Immune checkpoints refer to a plethora of inhibitory pathways in the immune system that are crucial for maintaining self-tolerance and modulating the duration and amplitude of physiological immune responses in peripheral tissues. It is now clear that tumors co-opt certain immune checkpoint pathways as a major mechanism of immune resistance, particularly against T cells that are specific for tumor antigens.

	T cell responses are regulated via multiple co-stimulatory and inhibitory interactions. T cell response to antigen is mediated by peptide-major histocompatibility complex (MHC) recognized by the T cell receptor. The B7 family of membrane-bound ligands binds both co-stimulatory and inhibitory receptors. Targeting cytotoxic T-lymphocyte-associated antigen (CTLA)-4 and programmed cell death ligand-1 (PD-L1) inhibitory receptors has been a major clinical focus.

	In 2011, the FDA approved the first immune checkpoint inhibitor, ipilimumab, an anti-cytotoxic T-lymphocyte-associated antigen (CTLA)-4 antibody for the treatment of metastatic and non-resectable melanomas.

 In 2015, pembrolizumab (marketed as Keytruda®) and nivolumab (marketed as Opdivo®) were the first of the anti-programmed cell death (PD)-1 pathway family of checkpoint inhibitors to gain accelerated approval from the FDA for the treatment of ipilimumab-refractory melanoma.

 Pembrolizumab has also been approved as a single agent for the first-line treatment of patients with metastatic non-small-cell lung cancer (NSCLC) whose tumors have high programmed cell death ligand-1 (PD-L1) expression, and nivolumab has also been approved for patients with metastatic squamous NSCLC who have progressed on or after platinum-based chemotherapy.

Combining Cancer Vaccines with Immune Checkpoint Inhibitors

	While immune checkpoint inhibitors represent a turning point in cancer immunotherapy, only about 10 to 50 percent of cancer patients with certain types of solid tumors have shown responses to treatments with immune checkpoint inhibitors. This has been attributed to properties of the tumor microenvironment, as well as a lack of tumor-infiltrating effector T cells.

	On the other hand, cancer vaccines serve to enlarge the pool of tumor-specific T cells, as well as to activate tumor-specific T cells that are dormant.

 While cancer vaccines have been shown to induce effector T cell infiltration into tumors, they need to overcome immune evasion to be fully effective.

	Consequently, it has been suggested that combining an immune checkpoint inhibitor with a cancer vaccine may result in a synergistic effect on the anti-tumor immune response. This hypothesis has been supposed by multiple preclinical studies. Supported by data from those studies, a clinical trial to test the combination of GM-CSF-secreting cancer vaccine (GVAX) and nivolumab in patients with metastatic pancreatic cancer has been initiated (ClinicalTrials.gov identifier: NCT02243371). Another clinical trial testing the combination of GVAX and nivolumab as neoadjuvant and adjuvant therapies for resectable pancreatic cancer (ClinicalTrials.gov identified: NCT02451982) is currently recruiting participants, as well.


	Cancer vaccine-based immunotherapy may help overcome the resistance of certain tumors to immune checkpoint inhibitors, while immune checkpoint inhibitors may enhance the efficacy of cancer vaccine therapies. This hypothesis remains to be proven in phase III clinical trials, but combination therapy with cancer vaccines and immune checkpoint inhibitors may be the next breakthrough in cancer therapy.

 Kleponis J, Skelton R, Zheng L. Fueling the engine and releasing the break: combinational therapy of cancer vaccines and immune checkpoint inhibitors. Cancer Biol Med. 2015;12(3):201-208.

 National Cancer Institute. FDA Approval for Sipuleucel-T. Available at https://www.cancer.gov/about-cancer/treatment/drugs/fda-sipuleucel-T.

 Kantoff PW, et al. Sipuleucel-T immunotherapy for castration-resistant prostate cancer. N Engl J Med. 2010;363:411-422.

 U.S. Food and Drug Administration. FDA approves first-of-its-kind product for the treatment of melanoma, October 27, 2015. Available at 

https://www.fda.gov/newsevents/newsroom/pressannouncements/ucm469571.htm

 Rehman H, et al. Into the clinic: Talimogene laherparpvec (T-VEC), a first-in-class intratumoral oncolytic viral therapy.

Andtbacka RHI, et al. Talimogene laherparepvec improves durable response rate in patients with advanced melanoma. J Clin Oncol. 2015;33(25):2780-2788.

 Fukuhara H, Ino Y, Todo T. Oncolytic virus therapy: A new era of cancer treatment at dawn. Cancer Sci. 2016;107:1373-1379.

 Pardoll DM. The blockade of immune checkpoints in cancer immunotherapy. Nat Rev Cancer. 2012;12:252-264.

 Hodi FS, et al. Improved survival with ipilimumab in patients with metastatic melanoma. N Engl J Med 2010;363:711-723.

 Mahoney KM, Freeman GJ, McDermott DF. The next immune-checkpoint inhibitors: PD-1/PD-L1 blockade in melanoma. Clin Ther. 2015;37(4):764-782.

 Brahmer J, et al. Nivolumab versus docetaxel in advanced squamous non-small-cell lung cancer. N Engl J Med 2015;373:123-135.

 van der Burg SH, et al. Vaccines for established cancer: overcoming the challenges posed by immune evasion. Nature Reviews Cancer. 2016;16:219-233.

 is the Senior Medical Director for Medical Services, Premier Research

Cancer vaccine-based immunotherapy may help overcome the resistance of certain tumors to immune checkpoint inhibitors, while immune checkpoint inhibitors may enhance the efficacy of cancer vaccine therapies.

Nina Baluja, M.D.

Articles

Cancer Immunotherapy Biomarkers: Four Trends to Watch

Vaccine-Induced Specific Immune Response Offers an Avenue to Potentiate Responses to Immune Checkpoint Inhibitor Therapy